Antibodies to human carcinoma antigen

ABSTRACT

A glycoprotein antigen which is generally characteristic of human carcinomas, regardless of the tissue associated with the carcinoma--human carcinoma antigen (HCA)--and which is generally not present on normal human cells. Immunodeterminant-containing fragments of HCA substantially separated from elements of HCA&#39;s naturally occurring environment are also disclosed. HCA is generally characterized by: a) a molecular weight in excess of 750,000; b) carbohydrate moieties characteristic of mucin-type glycoproteins and comprising a relatively high proportion of sialic acid, galactose, and N-acetylgalactosamine residues (e.g., at least 50% of the carbohydrate residues are sialic acid, galactose, or N-acetylgalactosamine residues); c) an isoelectric point below pH 3.0; d) presence generally on human carcinoma cells; e) absence generally from non-transformed human cells; f) at least one immunodeterminant that specifically reacts with anti-murine epiglycanin antibody AE3; and g) general insolubility in aqueous fluids (e.g., a phosphoric acid or an HCl solution) below pH 2.0 Also disclosed are antibodies to HCA, immunoassays for HCA, in vivo imaging using HCA-binding antibodies and therapeutics using HCA or anti-idiotypic HCA antibodies.

This is a divisional of application Ser. No. 08/192,840, filed Feb. 7,1994, now U.S. Pat. No. 5,545,532, which is a continuation-in-part ofapplication Ser. No. 08/014,450, filed Feb. 5, 1993, which is nowabandoned.

BACKGROUND OF THE INVENTION

This invention relates to antigen markers characteristic of tumors,antibodies to those markers, and immunoassays based on those markers. Italso relates to vaccines and to tumor imaging and immunotherapy usingtumor specific reagents.

When exposed to foreign substances, the immune system produces proteins,known as antibodies, which specifically bind to those foreignsubstances. The substances which trigger antibody production, and towhich antibodies bind, are known as antigens.

Antigens present on the surface of cells may serve as cell-surfacemarkers, and the presence of such markers can be detected by antibodiesthat are specific for them. In that way antibodies are used to detectthe presence of cells. Since cells can be characterized by the markersthey possess, it is possible to identify cell types using antibodies.

Many cell surface markers are glycosylated proteins (glycoproteins).Notable among the glycoproteins are mucin-type glycoproteins, which arelarge molecules with a high proportion of carbohydrate chains that areO-linked through N-acetylgalactosamine to serine and/or threonine in theprotein core.

There are various reports of antigens, some of which are mucin-typeglycoproteins, that appear on the surfaces of tumor cells but not on thesurfaces of normal cells of the same host. There are also variousdisclosures related to the use of such tumor specific antigens andantibodies to these antigens.

For example, there is a significant body of literature reporting studieson a sialomucin known as epiglycanin, which is found at the surface ofthe mouse carcinoma cell line TA3-Ha. Epiglycanin was first reported in1972 by Codington et al., (1972) Biochemistry 11:2559-2564. Mildproteolysis of viable TA3-Ha ascites cells with TPCK-trypsin, followedby fractionation of the released peptides by gel exclusionchromatography, gave a peak of glycopeptide material of high molecularweight, and that material has been the subject of numerous subsequentstudies: Codington et al., (1973) J. Nat'l Cancer Inst. 51:585-591;Slayter et al., (1973) J. Biol. Chem. 248:3405-3410; Codington et al.,(1975) Biochemistry 14:855-859; Codington et al., (1979) J. Nat'l CancerInstit. 63:153-162; Watkins et al., (1990) Carbohydr. Res. 213:185-200;Henningson et al., (1987) Cancer Immunol. Immunother. 25:231-241.Epiglycanin is generally characterized as having a molecular weight of500,000, about 80% of which is carbohydrate, largely Galβ (1→3) GalNAcside chains linked to serine or threonine residues in a polypeptidechain of about 1,300 amino acids. See, Van den Eijnden et al., (1979) J.Biol. Chem. 254:12153-12159. For reviews of this literature, seeCodington et al., (1992) Glycobiology 2:173-180; and Haavik et al.,(1992) Glycobiology 2:217-224.

Polyclonal antibodies to epiglycanin have been reported in Codington etal., (1984) J. Nat'l Cancer Instit. 73:1029-1038. Several monoclonalantibodies to epiglycanin have also been reported. Codington, U.S. Pat.No. 4,837,171; Haavik et al., (1992) Glycobiology 2(3):217-224. Thepolyclonal antibodies were found to react with a substance in peritonealor pleural fluid and in sera of patients with metastatic cancer.Codington et al., (1984) J. Nat'l Cancer Inst. 73:1029-1038.

Other tumor specific markers have been reported.

Samuel et al., in U.S. Pat. No. 5,110,911, report anadenocarcinoma-derived antigen which is shed by human tumor cells. Thisglycoprotein, termed Thomsen-Friedenreich (TF) antigen, has a molecularweight greater than 1,000,000 and is characterized by a non-crypticGalβ(1→3)GalNAc epitope.

Kortright, in U.S. Pat. Nos. 4,708,930 and 4,743,543, reports a murinemonoclonal antibody specific for an antigenic determinant on the surfaceor in the cytoplasm of human carcinoma cells and tissue. The antigenicdeterminant is designated "KC-4 antigen" and is said to appear in twoforms, one having a molecular weight of 480,000-510,000 and the secondhaving a molecular weight of 390,000-450,000. The KC-4 antigen wasdeveloped from human prostate adenocarcinoma.

Salem et al., in U.S. Pat. No. 4,921,789 report an antigen marker forhuman colorectal cancer, having a molecular weight of about 160,000. Theantigen is said to be non-reactive with antibodies to certain otherantigens.

O'Brien, in U.S. Pat. No. 4,921,790, reports a subunit of CA125 antigen,an antigen associated with cystadenocarcinoma of the ovary. The CA125antigen has a molecular weight of about 40,000.

Kufe, in U.S. Pat. Nos. 4,963,484 and 5,053,489, reports identification(by recombinant methods) of a peptide determinant of the DF3 antigenfrom human breast carcinoma.

Chu et al., in U.S. Pat. No. 4,939,240, report a monoclonal antibodysaid to reduce the mass of a human breast tumor xenograft in a mouse.

Hellstrom et al., in U.S. Pat. No. 4,918,164, report a monoclonalanti-idiotypic antibody having an antigen combining site related to thatof the p97 antigen of human melanoma.

Toth et al., in U.S. Pat. No. 4,914,021, report a monoclonal antibodyspecific for an antigen termed carcinoma or orosomucoid-related antigen(CORA). CORA is said to have a molecular weight of about 46,000-50,000,an isoelectric point of 3.0-3.5, and a carbohydrate content of 30% (byweight).

Yoshida, in U.S. Pat. No. 4,892,935, report an antibody to a humanpulmonary carcinoma antigen which reacts with human squamous cell lungcarcinoma but not with human small cell lung carcinoma.

Kjeldsen et al., (1988) Cancer Res. 48:2214-2220, report a breastcarcinoma glycoprotein termed TAG-72.

Springer et al., (1988) Carbohydr. Res. 178:271-292, report T and Tnhaptens in glycoproteins of human breast carcinoma.

Tjandra et al., (1988) Br. J. Surg. 75:811-817, report a breastcarcinoma glycoprotein termed MSA.

Ishida et al., (1989) Tumor Biol. 10:12-22, report a breast carcinomaantigen termed MFGM.

Lan et al., (1985) Cancer Res. 45:305-310, report a pancreatic carcinomaantigen termed DU-PAN-2.

Hanisch et al., (1988) Carbohydr. Res. 178:29-47, report an ovariancarcinoma antigen termed CA125.

Hinoda et al., (1988) Cancer J. 42:653-658, report a lung carcinomaantigen termed YH206.

SUMMARY OF THE INVENTION

We have discovered a glycoprotein antigen which is generallycharacteristic of human carcinomas, regardless of the epithelial tissueof origin. We call this antigen human carcinoma antigen (HCA). HCA isgenerally not present on normal human cells. One aspect of theinvention, in its broadest form, features HCA andimmunodeterminant-containing fragments of HCA substantially separatedfrom elements of HCA's natural environment. By immunodeterminants, wemean portions of HCA which are sufficient to bind to a monospecificanti-HCA monoclonal antibody or that are sufficient (alone or as ahapten conjugate) to raise an HCA-specific immune response in a mammal.We have found that HCA fragments having a molecular weight under about50,000 generally do not present suitable immunodeterminants. A substanceis substantially separated from elements of HCA's naturally occurringenvironment when it is sufficiently purified or isolated to be used inthe various methods of the invention discussed below, for example, to beused as an immunogen to raise anti-HCA antibodies or as a competitiveantigen in a competitive immunoassay. For example, the HCA in a washedimmunocomplex of HCA and HCA-binding antibody (such as would be producedin performing an immunoassay (competitive or ELISA) that uses anon-radioactive label) may be considered as substantially separated fromHCA's natural environment.

HCA is generally characterized by:

a) a molecular weight in excess of 750,000;

b) carbohydrate moieties characteristic of mucin-type glycoproteins andcomprising a relatively high proportion of sialic acid, galactose, andN-acetylgalactosamine residues (i.e., the sum of the weight of thosethree residues makes up a high proportion (e.g., at least 60%) by weightof total carbohydrate content);

c) an isoelectric point below pH 3.0;

d) presence generally on human carcinoma cells;

e) absence generally from non-transformed human cells;

f) at least one immunodeterminant that specifically reacts withanti-murine epiglycanin antibody AE3 (produced by hybridoma HAE-3,deposited with the American Tissue Type Culture Collection (ATCC),Rockville, MD under accession no. HB-9467); and

g) general insolubility in aqueous fluids (e.g., a phosphoric acid or anHCl solution) below pH 2.0;

More typically, HCA has a molecular weight over 1 million and anisoelectric point less than pH 2.5. HCA is generally found on tumorcells, for example on cells from colon, lung, pancreatic, mammary,prostate, and ovarian tumors of epithelial origin.

HCA may be generally characterized by affinity for anti-mouseepiglycanin antibodies. Affinity for some such antibodies may bedependent on the presence of an immunodeterminant that is sensitive to(i.e., immunoreactivity is substantially reduced or destroyed by)O-glycanase, periodate, or both, and affinity for some such antibodiesmay be enhanced by desialylation of HCA. HCA is also characterized bythe presence of at least 50% by weight carbohydrate.

HCA may also be characterized based on monosaccharide composition. HCAcomprises fucose, N-acetylgalactosamine, N-acetylglucosamine, galactose,mannose, and N-acetyl neuraminic acid, preferably in the followingamounts (±10%-40%), as determined by hydrolysis of HCA usingtrifluoroacetic acid, followed by determination of monosaccharidecomposition by HPAEC-PAD: fucose, 3.6%; N-acetylgalactosamine, 15.2%;N-acetylglucosamine, 11.2%; galactose, 27.8%; mannose, 25.6%; N-acetylneuraminic acid, 16.6%.

Amino acid composition may also be used to characterize HCA. HCAcomprises each of the following amino acid residues: Serine, Threonine,Glutamine, Asparagine, Leucine, Alanine, Glycine, Valine, Proline,Lysine, Isoleucine, Arginine, Phenylalanine, Tyrosine, Histidine,Cysteine. Each amino acid, Proline, Phenylalanine, Tyrosine or Cysteine,comprises 0-5% of the total amino acid residues of HCA. Preferably, theamino acids are present in the following amounts (±20%) as determined byhydrolysis of HCA using 6M HCl in vacuo, followed by amino acidanalysis: Serine, 10.2%; Threonine, 3.6%; Glutamine, 14.6%; Asparagine,8.2%; Leucine, 14.5%; Alanine, 6.6%; Glycine, 23.4%; Valine, 3.7%;Proline, <1.0%; Lysine, 4.8%; Isoleucine, 3.7%; Arginine, 4.3%;Phenylalanine, <1.0%; Tyrosine, <1.0%; Histidine, 2.4%; Cysteine, <1.0%;

HCA is also characterized by a buoyant density of 1.3-1.45 g/ml,preferably 1.34-1.41 g/ml, as determined by centrifugation in 47% cesiumtrifluoroacetate in accordance with the procedure described in Example19. Ability to bind to epiglycanin-specific antibodies, e.g., AE3, AD7,and BF11, can also be used to characterize HCA. HCA may be characterizedby the ability to bind to antibody AE3 with an affinity that is at least100× the affinity for antibody 49H.8.

Our discovery of HCA enables other aspects of the invention describedbelow.

A second aspect of the invention generally features antibodies specificfor human carcinoma antigen, in which the antibody is less reactive withmouse epiglycanin than with HCA. In preferred embodiments, the antibodyis raised by challenging the animal with HCA or an immunodeterminantcontaining a fragment thereof. It is also possible to raise HCA bindingantibodies by challenging an animal with anti-idiotypic antibodiesdescribed below. Preferably, the antibody requires a Galβ(1→3)GalNAcchain for binding. Also preferably, the antibodies used in the inventionare monoclonal antibodies.

In a third aspect, the invention generally features immunoassays todetermine the presence of HCA in a biological sample by reacting thesample with an antibody that binds to HCA, preferably the monodonalantibody, AE3, the reaction being carried out for a time and underconditions allowing the formation of an immunocomplex between theantibody and HCA. The quantitative determination of such animmunocomplex is then performed. In one version of the third aspect ofthe invention, the antibody used is an antibody generated byadministering to a mammal (e.g., a rabbit, goat, mouse, pig, etc.) animmunogen that is HCA, an immunogenic fragment of HCA, or ananti-HCA-binding idiotypic antibody. Other versions of this aspect ofthe invention feature the use of HCA-binding antibodies generally(regardless of whether they are raised to one of the immunogensdescribed above). For example a second version features a format inwhich immunocomplex formation is determined by competitive immunoassayprocedures--i.e., the HCA-binding antibody is reacted with sample andwith a competing antigen, e.g., a labeled tracer antigen or animmobilized competing antigen. A third version features a sandwichimmunoassay format which uses a second antibody that also binds HCA, oneof the two antibodies being immobilized and the other being labeled.

Preferred embodiments of the first version of the third aspect of theinvention feature detecting an immobilized complex between HCA and anHCA-binding antibody using a second antibody that is labeled and bindsto the first antibody. Alternatively, the first version features asandwich format in which the second antibody also binds HCA. In thesandwich immunoassay procedures, HCA-binding antibody can be a captureantibody attached to an insoluble material and the second HCA-bindingantibody can be a labeling antibody.

The above-described competitive immunoassay procedures or sandwichimmunoassay procedures can be used with the antibodies described abovein connection with the first version of the third aspect of theinvention. Suitable competing antigens to be used in competitive formatsfor the third aspect of the invention include: HCA; immunodeterminantcontaining fragments of HCA; epiglycanin; or immunodeterminantcontaining fragments of epiglycanin. One preferred competing antigen isa fraction of epiglycanin that does not bind to peanut lectin. Thecompeting antigen may be labeled or immobilized.

The assays of the third aspect of the invention can be used to determineHCA in samples including urine, plasma, serum, peritoneal fluid orlymphatic fluid, or solid tissue biopsies, such as breast carcinoma.This aspect of the invention also features immunoassay kits fordetecting human carcinoma antigen, comprising HCA-binding antibody andthe means for determining binding of the antibody to HCA in a biologicalsample. In preferred embodiments, the kit includes one of the secondantibodies or the competing antigens described above.

A fourth aspect of the invention features methods of imaging tumors(preferably in vivo) by administering an HCA-binding antibody bound(coupled) to an imaging agent such as a radioisotope or other imagingagent.

A fifth aspect of the invention features reagents and methods forselectively labeling human tumor tissue using labeled HCA-bindingantibodies bound (coupled) to detectable labels, such as enzymaticlabels, fluorescent labels, stains or radioisotopes.

A sixth aspect of the invention features treating human tumors, such asbreast carcinomas, by administering HCA-binding antibodies bound(coupled) to cytotoxins, such as chemotoxins (e.g., ricin) or cytotoxicradioisotopes.

A seventh aspect of the invention features HCA binding anti-idiotypicantibodies, which can be used as: a) vaccines protecting againstcarcinomas; b) immunotherapeutics to induce a cancer patient's immunesystem to generate an anti-carcinoma immune response; c) competingantigens in the above described competitive immunoassay formats; d) as apurification reagent to separate HCA-binding substances from non-HCAbinding substances.

An eighth aspect of the invention features HCA formulated in acomposition suitable for administration to humans, e.g., as a protectivevaccine or as an immunotherapeutic to induce a cancer patient's immunesystem to generate an anti-carcinoma immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph related to Example 2 concerning HCA purification.

FIG. 2 is a graph related to Example 3 concerning HCA purification.

FIG. 3 is a graph related to Example 5 concerning fragmentation of HCA.

FIG. 4 is a graph related to Example 6 concerning purification of HCAfragments.

FIG. 5 is a graph related to Example 10.

FIG. 6 is a graph related to Example 12.

FIG. 7 is a graph related to Example 13.

FIG. 8 is a graph related to Example 14.

FIG. 9 is a graph related to Example 15.

FIGS. 10-12 are graphs related to Example 16.

FIGS. 13 and 14 are graphs related to Example 19.

FIG. 15 is a graph related to Example 20.

FIGS. 16A and 16B are graphs related to Example 21.

FIGS. 17 and 18 are graphs related to Example 22.

FIG. 19 is a graph related to Example 23.

FIGS. 20 and 21 are graphs related to Example 24.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Sources of HCA

As described above, HCA is a human carcinoma antigen and can generallybe obtained by culturing established human carcinoma cell lines, or fromascites fluid of carcinoma patients, particularly breast and ovariancarcinoma patients. Appropriate cell lines can be screened for HCAproduction using the immunoassays described below. Preferred cell linesare those producing relatively higher concentrations of HCA asdetermined by such assays.

One cell line source of HCA is the human endometrial carcinoma cell lineKLE (ATCC accession no. CRL1622) described in Richardson et al.Gynecologic Oncology 17:213-230 (1984). Those skilled in the art willappreciate that there are numerous other possible cell line sources. Forexample, the ATCC has numerous carcinoma cell lines from various typesof carcinomas, including KLE, described above.

HCA can also be isolated from fluids (pleural, peritoneal, serum, etc.)of patients with carcinomas including carcinomas of the breast, colon,prostate, ovary, lung, etc.

II. Purification of HCA

In general, the carcinoma cell line is cultured (for example, asdescribed by Richardson et al.), and the spent medium is concentrated,e.g. using Filtron Omega Ultrasette filtration system equipped with a100K membrane, and its high-molecular weight fraction is isolated. HCAimmunoreactivity can be followed using an anti-HCA antibody describedbelow, or using anti-epiglycanin antibodies such as AE-3, describedabove and reported in Codington U.S. Pat. No. 4,837,171. The substantialmajority of the HCA immunoreactivity is retained in the high molecularweight fraction.

The high molecular weight fraction is subjected to furtherchromatography, e.g. Sepharose CL-2B. Again, the majority of the HCAimmunoreactivity will be in the highest molecular weight range (1-2million molecular weight)

Further chromatography (e.g. Mono P coupled to a Pharmacia FPLC system)is performed on the resulting high molecular weight fraction. HCAactivity is followed by immunoassay, e.g., with an enzyme linkedcompetitive binding assay. HCA tends to precipitate on the Mono P columnas an insoluble precipitate that is not effectively removed by NaClgradient. This precipitate is substantially insoluble in solutions ofurea, dithiothreitol, guanidine hydrochloride, or perchloric acid.

III. Obtaining Immunodeterminant Containing HCA Fragments

Because of the extraordinary size and insolubility of native HCA, it isusually convenient to work with immunodeterminant-containing HCAfragments. Such fragments are prepared by partial proteolyticdegradation (e.g. using an enzyme such as TPCK-trypsin, Pronase™, orpepsin). The digest can be purified by chromatography, e.g. gelexclusion, collecting fractions having HCA immunoreactivity.

IV. Antibodies To HCA

HCA recovered as described above, or fragments of it, can be used as animmunogen to challenge a mammal, e.g. a mouse, rabbit, rat or goat.Those skilled in the art will recognize that various protocols may beused. Preferably, protocols used in producing anti-epiglycaninantibodies (described below) can be used to make anti-HCA monoclonalantibodies. Preferably the animal is challenged over a substantialperiod (e.g. 5 months). See generally, Haavik et al., (1992)Glycobiology 2:217-224. For mouse monoclonal antibodies, standard fusionpartners may be used to immortalize spleen cells from the challengedanimal. See, e.g., Kohler and Milstein, (1977) Nature 256:495-497. Theresulting anti-HCA monoclonal antibodies or polyclonal antibodies may beused in the various immunological procedures described below.

V. Immunoassays For HCA

Immunoassays for HCA can be performed using the anti-HCA antibodiesdescribed above or other HCA-binding antibodies such as anti-epiglycaninantibodies that bind HCA. In general, any suitable immunoassay formatcan be used. For example, an immobilized HCA/anti-HCA antibody complexcan be formed (by radioimmunopreciptation) and detected with antibodyspecific for the anti-HCA antibody. Thus, if the HCA-binding antibody isa mouse-generated antibody, the immunocomplex may be detected with alabeled goat anti-mouse antibody. We use the term labeling generally todescribe all types of well known labels such as enzymes (e.g.,horseradish peroxidase) or other suitable labels, including radiolabels,fluorophores, and chromophores. Biotin/avidin labeling systems may beused.

In a competitive format (e.g. as described in example 18) it is possibleto use a competing antigen that is labeled or immobilized. It isparticularly important in a competitive format to use a suitablecompeting antigen. One preferred competing antigen features a fractionof epiglycanin known as epiglycanin-A, obtained as described below.(Example 17)

Best results are obtained by the addition of serum first, thenmonoclonal antibody, to the wells. Less satisfactory results areobtained if the two components are mixed prior to their addition to thewells. It is also important that the addition of serum and antibody becompleted within a short period of time (i.e., about 30-50 min).

The PBS used in this competitive assay is at pH 7.5-7.6 and containsthree times the concentration of phosphate that is generally used inimmunoassays, in order to minimize variability in the assay due to pHchanges.

When performed under optimal conditions, the competitive binding assaymay be used to quantitatively determine the concentration of HCA presentin the blood (serum or plasma) of individuals with carcinomas. Thepreferred procedure embodies the following steps.

The wells of a 96 well microtiter immunoplate are coated by adding 100μl of a solution of Epiglycanin A in PBS (7.5 ng/ml) and allowing thissolution to stand for 15-20 hours at 4° C. Alternatively, the coatingsolution may be allowed to dry in the wells. Such plates may be storedin the dry state for several weeks at 4° C. After discarding the coatingsolution, the wells are blocked with Superblock (Pierce Chemical Co.,275 μl/well) for 60 min. at 4° C. They are washed with PBS containingTween 20 (0.05%), a solution used for all washes. The addition of 50 μlof sample to be assayed (i.e., serum at full strength, HCA standards,other HCA-containing material, or controls) is quickly followed by theaddition of 50 μl of antibody. The optimal concentration range for HCAis approximately 1-200 ng/ml. Monoclonal antibody is used at aconcentration of 200 ng/ml (10 ng/well). All dilutions are made in aSuperblock solution, and each sample is assayed in triplicate.

The plate is incubated for 14-20 hours, then washed as before. After theaddition of 100 μl of goat anti-mouse IgM, coupled to horseradishperoxidase (1:3000 in PBS), the plate is incubated for 2.0 hours at 4°C. then washed. The concentration of HCA is correlated with the amountof bound antibody. Antibody concentration is determined by the additionof substrate 2,2',-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) at a concentration of 1 mg/ml citrate buffer!, with 1 μl/ml of30% H₂ O₂. Color develops at 20°-25° C. and is read using aspectrophotometer at 405 nm.

These assays can be used to: a) determine the presence of a carcinomainitially; b) to stage the carcinoma and thus provide guidance forfuture treatment (systemic versus localized treatment); c) to follow acourse of treatment of a carcinoma; and (d) determine recurrence of acarcinoma.

VI. In Vivo Imaging Using Anti-HCA Antibodies

Anti-HCA antibodies can be used to specifically image tumors, in vivo.For example, the antibodies can be covalently bound to radioisotopessuitable for in vivo imaging, such as ¹¹¹ In or ^(99m) Tc. Less favoredis ¹³¹ I. Those skilled in the art will understand that there arenumerous well known techniques for generating the radioisotopes at issueand binding the radioisotopes to antibodies. See, for example,Bartorelli, U.S. Pat. No. 4,732,862 hereby incorporated by referenceregarding radioisotope labeling of antibodies. See also, Goldberg etal., (1978) New England J. Med. 298:1384 et seq.; Seragini et al.,(1989) Clin. Nucl. Med. 14:580-587; and Surwit, (1990) Antibody,Immunoconjugates, and Radiopharmaceuticals 3:48-49 regarding preparationof radiolabeled antibodies. Kits for radiolabeling can be obtained,e.g., from various commercial sources, such as Amersham, Mediphysics,Syncor, or Mallinckrodt.

Those skilled in the art will also understand that the imaging agentdescribed above can be administered (infused) by various well knowntechniques, such as injection at a specific site or by i.v. infusion.See, e.g., Treves, U.S. Pat. No. 4,729,380. Readings are taken bystandard techniques such as a body scanning scintigram using a gammacamera interfaced with a computer.

VII. Tissue Staining Using HCA-Binding Antibodies

Anti-HCA antibodies or HCA-binding antibodies can be coupled (bound) toimaging agents such as the radioisotopes described above or otherimaging agents such as fluorescent imaging agents, and visual imagingagents (stains). See, e.g., Kortright U.S. Pat. No. 4,708,930, citedabove. Standard techniques are used to obtain tissue sections and tolabel them with HCA-binding antibodies that are coupled to anappropriate label. Another technique involves the use of electronmicroscopy (electron dense) imaging agents (e.g., gold particles).

VIII. Cell-Selective Immunotherapeutics

To destroy the tumor cells, HCA-binding antibodies are coupled tocytotoxic agents such as ricin A chain, abrin A-chain, modeccin A-chain,gelonin, melphalan, bleomycin, adriamycin, daunomycin, or pokeweedantiviral proteins (PAP, PAPII, PAP-S). Those skilled in the art willrealize that there are numerous radioisotopes or chemocytotoxic agentsthat can be coupled to tumor specific antibodies by well knowntechniques, and delivered to specifically destroy tumor tissue. See,e.g., Blattler et al. U.S. Pat. No. 4,542,225.

IX. Anti-idiotypic Antibodies

Those skilled in the art will realize that known techniques can be usedto raise anti-idiotypic antibodies, e.g., idiotypic antibodies that willbind to HCA-binding antibodies. HCA-binding anti-epiglycanin antibodiesor anti-HCA antibodies can be used as immunogens to challenge a mammalaccording to the immunization regime described above for anti-HCAantibodies. See. e.g., Hellstrom et al., in U.S. Pat. No. 4,918,164,cited above. The resulting anti-idiotypic antibodies can be used as: a)vaccines to induce an immune response resulting in protection againstthe development of carcinomas; b) immunotherapeutics to induce a cancerpatient's immune response to his carcinoma; c) competing antigens in theabove described competitive immunoassay formats; d) as purificationreagents to separate HCA-binding substances from non-HCA bindingsubstances.

For in vivo applications, those skilled in the art will recognize thatthere are various known techniques to make mouse monoclonal antibodiesmore compatible with human therapies, e.g., Winter et al., (1991) Nature349:293-299; Sahagan et al., (1986) J. Immunol. 137:1066-1074; and Bosset al., U.S. Pat. No. 4,816,397.

IX. HCA-Based Vaccines And Immunotherapeutics

Those skilled in the art will recognize that HCA (or immunodeterminantfragments of it) purified as described above, can be formulated as avaccine, using standard vehicles such as pyrogen free buffered saline.The vaccine can be administered by standard techniques (e.g., i.v.) overa suitable course of treatment to induce an anti-carcinoma immuneresponse. Such a response can be prophylactic or therapeutic (forpatients already diagnosed as having a carcinoma).

X. Specific Examples

The following specific experiments are provided to illustrate theinvention, without limiting its scope.

EXAMPLE 1 Filtration of Spent Medium From Cultured Carcinoma Cell Line

The KLE endometrial carcinoma cell line referenced above was cultured asgenerally described by Richardson et al. (1984). Generally, the cellswere grown in medium containing equal volumes of Ham F12 and Dulbecco'sModified Eagle's Medium with 15% fetal calf serum (GIBCO), 60 mU/mLinsulin, 20 U/mL penicillin, 20 μg/mL streptomycin and 0.5 μg/mlamphotericin. Samples of spent medium from cultures with confluent cellswere withdrawn and spun. The spent medium was concentrated by a FiltronOmega Ultrasette, equipped with a 100K membrane (4° C.), and the highmolecular weight fraction was tested for HC-antigen content by theenzyme competitive binding assay, as described below.

EXAMPLE 2 Gel Filtration Chromatography on a Sepharose CL-2B Column

Samples of concentrated medium described in Example 1 were subjected togel filtration chromatography on a column (5×180 cm) of Sepharose Cl-2Beluted with PBS buffer pH 7.50. Fractions were collected and assayed forcarbohydrate by the general phenol-sulfuric acid method of Dubois et al.(1956) Anal. Chem. 28.:350-356, and the absorbance was read at 490 nm.The collected fractions were assayed for HC-antigen content using theenzyme competitive binding assay described below. After 10chromatographic runs (of 3 days each) fractions were pooled. Fraction Awas concentrated by the Filtron Ultrasette with an Omega 100 K membraneand kept stored at -20° C. until it was subjected to furtherpurification and characterization.

Specifically, FIG. 1 shows the results of chromatography on two coupledcolumns of Sepharose CL-2B (5×90 cm each). Fractions were tested forreactivity to HCA-binding antibodies, and for protein and carbohydratecontent as described below. Elution volumes of molecular weight markersare indicated by arrows: 1: Dextran T 2000 (2,000,000); 2: Thyroglobulin(670,000); 3: Bovine serum albumin (BSA) (67,000); 4: K₂ Cr2O₇ (294).Fractions A, B, C and D were pooled as indicated.

EXAMPLE 3 FPLC Ion Exchange Chromatography on Mono P Column

Samples of fraction A from the Sepharose CL-2B column (Example 2) wereapplied to a column of Mono P (Pharmacia) (10×300 mm) coupled to aPharmacia FPLC system. The column was eluted with a gradient of sodiumchloride in 15 mM phosphate buffer pH 7.20. The absorbance at 280 nm ofthe eluate was monitored and fractions collected and tested for activitybinding to HCA-binding antibodies by the enzyme competitive bindingassay as described below.

FIG. 2 shows the absorbance of the effluent.

EXAMPLE 4 Redissolving HCA

Samples of suspended precipitate of Fr. A (Example 3) were incubatedwith either 2M urea, 4M urea, 2% dithiothreiothol, 2% EDTA, 4M guanidinehydrochloride for 16 h at room temperature. The samples were then testedfor binding activity to HCA-binding antibodies by the enzyme competitivebinding assays as described below. Substantially no HCA activity wasfound in the solutions.

EXAMPLE 5 Fragmentation of HCA

Suspensions of Fr. 1 (Example 3) were incubated with 20 μg/mLTPCK-trypsin in 0.1M sodium phosphate buffer pH 6.80 for 16 h at 37° C.The incubation mixtures were then applied to a Superdex 200 (10×600 mm)column and eluted 1.0 mL/min with 0.1M sodium phosphate buffercontaining 0.15M NaCl. The absorbance at 280 nm was monitored, andfractions were-collected and tested for binding activity to HCA-bindingantibodies as described below. FIG. 3 depicts the absorbance of theeffluent at 280 nm. Elution volumes of molecular-weight-markers areindicated: 1: Dextran T 500 (500,000); 2: Thyroglobulin (670,000); 3:Aldolase (158,000); 4: Bovine serum albumin (BSA) (67,000); 5:Ribonuclease A (13,700).

EXAMPLE 6 Affinity Chromatography on a Column of Peanut AgglutininSepharose

A sample of Fr. 1 from the Superdex 200 column (Example 5) was appliedon a column (10×50 mm) of Arachis hypogea lectin insolubilized on 4%beaded agarose (Sigma). The column was eluted at 0.1 mL/min with: a)0.1M Naphosphate buffer pH 6.8 containing 0.2M NaCl; b) a gradient of0-2M methyl-β-D-galactopyranoside in 0.1M Na-phosphate buffer pH 6.8containing 0.2M NaCl. The absorbance at 280 nm was monitored, fractionscollected and tested for binding activity to HCA-binding antibodies bythe enzyme biotin sandwich assay as described below. FIG. 4 depictsabsorbance of the effluent at 280 nm.

EXAMPLE 7 Evaluation of HCA Epitopes

Samples of Fr. A from the Sepharose CL-2B column of the spent KLE medium(Example 1) were subjected to either periodate oxidation (10 mM NalO₄ atpH 4.0 for 30 min at 20° C.), incubation at 37° C. for 16 h withTPCK-trypsin, neuraminidase (Vibrio cholera, type II), orendo-α-N-acetyl-D-galactosaminidase (O-Glycanase). The samples were thendiluted and tested for HC-activity by the enzyme competitive bindingassay. The above reagents altered binding activity for an HCA-bindingantibody as follows: Neuraminidase 19%--activity remaining;endo-N-acetyl-galactosaminidase--activity remaining 0.1%; periodate(10mM, 20° C., 1 h)--activity remaining 10%; TPCK-trypsin--activityremaining 137%.

EXAMPLE 8 Enzyme Competitive Binding Assay For Non-Serum Samples

To the wells of a Nunc Maxisorp 96 well plate were added 100 μl ofepiglycanin (50 ng/mL) in PBS, pH 7.50, and incubated for 16 h at 4° C.The wells were blocked by incubation for 45 min with 0.5% BSA in PBS, pH7.50 (PBS-BSA). Two solutions were added: a) 50 μL of a standardsolution of epiglycanin (1-200 ng/ml) or sample; and b) 50 μL ofpurified monoclonal anti-epiglycanin IgM (dilution 1: OD₂₈₀ ×10,000).Both antibody and antigen were diluted with PBS-BSA. The mixture wasincubated on a shaker for 16 h at 20° C. The wells were washed threetimes (Skatron microplate washer) with PBS containing 0.05% Tween 20 andincubated for 2 h at 20° C. with alkaline phosphatase labeled goatanti-mouse IgM dissolved in PBS-BSA. The wells were washed three timesand then 100 μL substrate solution containing 1 mg/mL p-nitrophenylphosphate in 0.1M ethanolamine pH 10.0 was added. The absorbance wasread at 405 nm in a Bio-Rad Microplate reader coupled to a Macintosh SEcomputer using the Microplate Manager TM program.

EXAMPLE 9 Enzyme Biotin Sandwich Assay

To the wells of a Nunc Maxisorp U 96 microtiterplate were added 100 μLmonoclonal anti-EPGN antibody (2 μg/mL) diluted in PBS pH 7.50 andincubated for 16h at 4° C. The coating solution was then removed and thewells were blocked by incubation for 45 min with 200 μL 0.5% BSA in PBS,pH 7.50 (PBS-BSA) at 4° C. After the blocking solution was removed, 100μL solutions of epiglycanin standard or samples were added to each well.After incubation for 2 hours at 20° C., they were emptied and washedtwice with 200 μL PBS pH 7.50 containing 0.05% Tween 20 (PBS/Tween)(Skatron Plate washer). 100 μL of a solution of biotinylatedanti-epiglycanin antibody (1 μg/ML) diluted in PBS-BSA was added to eachwell and the mixture incubated for 2 h at 20° C. The wells were emptiedand washed twice with 200 μL PBS/Tween. 100 μL of a solution ofstreptavidin-peroxidase diluted 1:5000 in PBS-BSA was added to each welland incubated for 2 hours at 20° C. The wells were emptied and werewashed three times with 200 μL PBS/Tween. 100 μL of a freshly preparedsolution containing 1 mg/mL O-phenylene diamine and 0.5 μL/mL H₂ O₂(30%) in citrate-phosphate buffer (pH 5.9) was added to each well. Theplate was incubated at 20° C. until appropriate color developed, afterwhich 50 μL 0.3M citric acid was added to the wells to stop thereaction. Absorbance was read at 490 nm in a Bio-Rad microplate readerusing the Microplate Manager TM program for calculation ofconcentrations.

EXAMPLE 10 Isoelectric Focusing

Isoelectric focusing of HCA isolated from the ascites fluid of a patientwith ovarian carcinoma was achieved as follows. Focusing was performedin Rototofor™ Cell (Bio-Rad) for 4 h at 12 W with Biolyte 3-10ampholyte.

Isoelectric focusing of HCA from cultured endometrial carcinoma celllines was performed in Rototofor™ Cell (Bio-Rad) for 4 h at 12 W withBiolyte 3-10 ampholyte. FIG. 5 depicts the results of this experiment.

EXAMPLE 11 Epiglycanin Monoclonal Antibodies

Anti-epiglycanin monoclonal antibodies were prepared as describedpreviously (Codington, U.S. Pat. No. 4,837,171; Haavik et al., 1992Glycobiology). Briefly C57BL/J mice were immunized by subcutaneousinjections of 10⁶ viable TA3-Ha ascites cells and boosted with purifiedepiglycanin. Spleen cells of the immunized mice were fused with BALB/c(NS1) mouse myeloma cells, as described by others. Hybridomas producingantibodies against epiglycanin were screened by an antibody captureassay using microtiterplates coated with purified epiglycanin. Theantibodies were purified as described previously (Haavik et al., 1992Glycobiology. One specific purification technique involves elution froman Avid Al affinity column (9×20 mm, Bio Probe) followed by successiveHPLC fractionations on an AB_(x) column (7.75×100 mm, J. T. Baker) and aProtein Pak™ DEAE 5PW column (7.5×75 mm, Waters).

EXAMPLE 12 HCA in Ascites

Ascites fluid from carcinoma patients was obtained by standardtechniques and measured with the competitive immunoassay describedbelow, using two anti-mouse epiglycanin monoclonal antibodies. Theresults are shown in FIG. 6. Results with one antibody are shown withsolid bars and with the other antibody are shown with a stippled bar. InFIG. 6 the patients had been diagnosed as follows: #1=breast carcinoma;#2=ovarian carcinoma; #3=breast carcinoma; and #4=ovarian carcinoma.

EXAMPLE 13 HCA from Ascites

HCA activity of ascites fluids of patient #4 of Example 12 and spentmedium from an endometrial carcinoma cell line were concentrated byFiltron Diaflo membrane with a molecular weight cutoff of 100,000. HCAactivity was tested using an anti-epiglycanin monoclonal antibody in acompetitive binding assay. The results are shown in FIG. 7. Most of theHCA activity was found in the fraction retained by the membrane. HCAactivity in the fraction which passed through the membrane was too lowto be visible on the graph shown in FIG. 7.

EXAMPLE 14 Further Purification and Fragmentation

The concentrated ascites fluid was subjected to Sepharose CL-2B gelfiltration and the material was concentrated using a Filtron Ultrasette(Filtron Technology Corporation, Northborough, Mass.) with a molecularweight cutoff limit of 100,000 Da. Specifically, the concentratedascites fluid was applied to a column of Sepharose CL-2B (5×180 cm)(previously equilibrated in phosphate buffered saline (PBS) (pH 7.50))at a rate of 1 ml/min. Fractions were collected and assayed forcarbohydrate by the phenol-sulfuric acid method (Dubois et al., 1956)and the absorbance read at 490 nm. The determination of the approximateprotein concentration in the chromatographic profiles was done bymeasurement of the absorbance at 280 nm in a UV-visible recordingspectrophotometer, model UV-160A from Shimadzu. The absorbance was readagainst PBS buffer as a reference. The collected fractions were assayedfor HC-antigen content using enzyme competitive binding assay with 4monoclonal anti-epiglycanin antibodies. The fractions were pooled after13 chromatographic runs. Fraction A was concentrated by the FiltronUltrasette with an Omega 100K membrane, and kept stored at -20° C. untilfurther purification.

The results are shown in FIG. 8. Elution volumes of molecular weightmarkers are indicated: 1: Dextran T 2000 (2,000,000); 2: Thyroglobulin(670,000); 3: Bovine serum albumin (67,000); 4: K2Cr207 (294). Thefractions were pooled as indicated.

EXAMPLE 15 Fragmentation and Further Column Chromatography

In order to digest the HCA, an equivalent volume of TPCK-trypsin (40μg/ml) in 0.1M sodium phosphate buffer pH 6.8 was added to an HCAsuspension and incubated for 16 h at 37° C.

The concentrated and trypsin-digested fraction A eluted from theSepharose CL-2B column was applied to a column of Superdex 200 HR 16/60column (10×600 mm) coupled to an HPLC-instrument. The column had beenequilibrated with 0.1M sodium phosphate buffer (pH 6.8) containing 0.2MNaCl, and was run at a flow rate of 1.0 ml/min. Fractions of 1.0 ml werecollected. The absorbance at 280 nm of the effluent was monitored andfractions collected and assayed for HC-antigen content by the enzymecompetitive binding assay as described above.

The results are described in FIG. 9. Elution volumes of molecular weightmarkers are indicated: 1: Dextran T 500 (500,000); 2: Thyroglobulin(670,000); 3: Aldolase (158,000); 4: Bovine serum albumin (67,000); 5:Ribonuclease A (13,700). The fractions were pooled as indicated.

EXAMPLE 16 pH-Dependent Precipitation of HCA from Human Ascites Fluid

FIGS. 10-12 depict turbidity of a stirred solution of HCA from ascitesfluids (Example 12), containing about 100 U/ml of HCA (equivalent inimmunoreactivity to 100 ng/ml epiglycanin). The solution pH was adjustedby dropwise addition of phosphoric acid. In FIG. 10., one minute aftereach addition, the absorbance was read at 500 nm. HCA precipitatedspontaneously around pH 1.1. In FIG. 11, the solution was adjusted to pH1.0 by addition of phosphoric acid, and measurements were taken over theperiod 5-30 min after that adjustment. NaOH was added to adjust to pH10.0 with stirring. Aliquots were withdrawn and absorbance was measured(FIG. 12).

EXAMPLE 17 Epiglycanin A

A preferred competitor for competitive HCA immunoassays is anepiglycanin fraction termed Epiglycanin A which is obtained by affinitychromatography (using immobilized peanut lectin) of ascites fluids ofmice bearing the TA3-MM/1 ascites cell. The fraction bound to the lectinis termed Epiglycanin B, and the fraction passing through the columnfreely is Epiglycanin A.

EXAMPLE 18 Competitive Binding Immunoassay

A competitive binding assay (CBA) particularly useful for serum sampleswas developed for the determination of the concentration of the HCA inhuman serum. The following steps are employed:

1. Coating. 100 μl of a solution of 25 ng/ml of Epiglycanin-A inphosphate buffered saline (PBS) at pH 7.6 and at 4° C. is incubated inwells for 8-16 hours.

2. Wells are washed after each incubation with PBS.

3. Blocking. A solution of human serum albumin (HSA) (0.6%) in PBS isincubated in the wells for 2 hours.

4. Serum is pre-treated (incubated) with a stabilizing solution (0.010Msodium borate) using a 1:5 sample dilution, at 4° C. for 4-12 hours.This solution is then diluted 1:5 with 0.1% HSA.

5. Monoclonal antibody is diluted with 0.1% HSA at a dilution ofapproximately 1:(O.D.₂₈₀ ×10,000).

6. To the washed (PBS) wells are added, in turn, 50 μl of serum (1:25,see 4. above) and 50 μl of diluted antibody (see 5. above).

7. The plate is covered with a plastic sheet and incubated with gentleshaking at 4° C. for 12-18 hours.

8. After washing the plate with PBS, 100 μl of goat anti-mouse IgM, (μchain specific), phosphatase labeled (1:4,000-10,000) is added, and theplate is incubated at 4° C. with gentle shaking for 3-4 hours.

9. To the washed wells are added 100 μl of substrate (15 mg ofparanitrophenyl phosphate in 12 ml of carbonate-bicarbonate buffer, pH10.3).

10. Color is allowed to develop at room temperature (20°-22° C.). Colorintensity is read at 405 nm in a Dynatech automatic plate reader.

EXAMPLE 19 Buoyant Density of HCA

The buoyant density of the HCA from ascites fluid and the KLE-1 cellline was determined by gradient centrifugation in cesiumtrifluoroacetate according to methods known in the art. Samples of HCAwere centrifuged in 47% cesium trifluoroacetate at 130,000× g for 72 hin a Beckmann T-1250 rotor. Fractions of 1 ml were collected, thedensity of each sample determined, and aliquots of each sample testedfor HCA activity by the enzyme competitive binding assay describedabove. The results of such a binding assay using HCA from ascites fluidof a patient with ovarian carcinoma and spent medium of the KLE-1 cellline are shown in FIGS. 13 and 14, respectively.

In FIG. 13, HCA purified from human ascites fluid (Fraction A from aSepharose CL-2B column in FIG. 8) was centrifuged at 130,000× g for 72 hin 47% cesium trifluoroacetate (O represents density). HCA-activity (▪)was tested in an enzyme competitive binding assay using anti-EPGN G-1antibody.

In FIG. 14, HCA purified from spent medium of a cultured endometrialcarcinoma cell line (KLE) was centrifuged at 130,000× g for 72 h in 47%cesium trifluoracetate (O represents density). HCA-activity (▪) wastested by an enzyme competitive binding assay using anti-EPGN B-4antibody.

EXAMPLE 20 Affinity Chromatography using Columns of ImmobilizedMonoclonal Anti-Epiglycanin Antibodies

To purify HCA, samples of spent medium from the KLE-1 cell line or fromhuman ascites fluid were applied to a column (1×5 cm) of Carbolink™Coupling Gel (Pierce Chemical, Rockford, Ill.) to which anti-epiglycaninAE-3 was covalently coupled. After application of the sample, the columnwas eluted with PBS pH (7.50), and then with PBS pH (7.50) containing 4Mguanidine-HCl. Fractions of 2 ml were collected, aliquots of thefractions diluted, and samples tested for HCA activity by the enzymecompetitive binding assay. A typical elution profile for HCA from spentmedium of the KLE-1 cell line is shown in FIG. 15.

FIG. 15 shows a chromatographic analysis of spent medium of the humanendometrial carcinoma cell line, KLE, on an affinity column ofanti-epiglycanin AE-3. Elution with 4 M guanidine-HCl was started asindicated by the arrow. The indicated peak fractions were pooled,dialyzed, subjected to SDS-PAGE, and blotted onto PVDF-membrane forfurther analysis.

EXAMPLE 21 HCA by SDS-PAGE and Visualization of the HCA on Blots

The peak fractions eluted from the affinity column with 4M guanidine-HClwere pooled, dialyzed extensively against distilled water, lyophilized,redissolved in sample buffer for SDS-PAGE containing mercaptoethanol,heated in a boiling water bath for 5 minutes and subjected to SDS-PAGEusing an 8% gel (Laemmli, (1970) Nature 227:680-685). Theelectrophoretically separated components were transferred to Immobilon™PVDF Transfer Membrane (Millipore) by electroblotting. The blots werestained for protein with Coomassie Brilliant Blue R-250 and, HCA wasdetected by incubation with anti-epiglycanin antibody AE-3, followed byincubation with affinity purified goat-anti mouse IgM conjugated toalkaline phosphatase (Boehringer Mannheim). Visualization of proteinbands was accomplished by incubation with a substrate mixture containingnaphthol ASMX phosphate and O-dianisidine, tetrazotized (Fast BlueB-salt) in 0.1M Tris-HCl buffer pH 10.0 and washing with distilled waterafter appropriate color development (See FIG. 16B).

FIGS. 16A and 16B show a Coomassie-stained SDS-PAGE gel 8%polyacrylamide) and a PVDF blot of HCA purified from KLE cells,respectively.

FIG. 16A: (A) Molecular weight standard: myosin (MW 212,000 Da),α-2-macroglobulin (Mw 170,000 Da), β-galactosidase (MW 116,000 Da),transferring (MW 76,000 Da); (B) Concentrated medium from the KLE cellline applied to affinity column with immobilized AE-3; (C) Material notbound to affinity column with immobilized AE-3; and (D) Fraction boundto AE-3 column and eluted with 4M guanidine-HCl.

FIG. 16B: (B) Concentrated medium from the KLE cell line applied toaffinity column with immobilized AE-3; and (C) Material not bound toaffinity column with immobilized AE-3; (D) Fraction bound to AE-3 columnand eluted with 4M guanidine-HCl.

EXAMPLE 22 Determination of Amino Acid and Monosaccharide Composition ofthe HCA

After staining with Coomassie Brilliant Blue, the HCA band was excisedfrom the PVDF-membrane. One part of the band was subjected to hydrolysisusing 6M HCl in vacuo followed by amino acid analysis of the hydrolysate(Table 1). Another part of the blot was subjected to hydrolysis using 2Mtrifluoroacetic acid followed by determination of monosaccharidecomposition by HPAEC-PAD (Weitzhandler, et al. (1993) J. Biol. Chem.268:5121-5130), (Table 2).

                  TABLE 1                                                         ______________________________________                                        Amino acid composition of affinity purified HCA                               from ascites fluid.                                                           Amino Acid   Residues/1000 Residues                                           ______________________________________                                        Serine       102                                                              Threonine    36                                                               Glutamine    146                                                              Asparagine   82                                                               Leucine      145                                                              Alanine      66                                                               Glycine      234                                                              Valine       37                                                               Proline      --                                                               Lysine       48                                                               Isoleucine   37                                                               Arginine     43                                                               Phenylalanine                                                                              traces                                                           Tyrosine     traces                                                           Histidine    24                                                               Cysteine     --                                                               ______________________________________                                    

The amino acid composition of the HCA was shown to be significantlydifferent from that of epiglycanin of the mouse mammary carcinoma cellline TA3-Ha (Codington and Haavik (1992) Glycobiology 2:173-180), MUC1(episialin) mouse tumor-associated mucin (Spicer, et al. (1991) J. Biol.Chem. 266:15099-15109.), the sialomucin ASGP-1 of the 13762 rat mammaryadenocarcinoma cell line (Carraway and Spielman (1986) Mol. Cell.Biochem. 72:109-120), TAG-72 from the human colon carcinoma zenograft,LS-174T (Sheer, et al. (1988) Cancer Res. 48:6811-6818), epitectin (Caantigen) isolated from human laryngeal carcinoma (Bhavanandan, et al.(1988) Ind. J. Biochem. Biophys. 25:36-42), or pancreatic tumor mucinfrom the human pancreatic tumor cell line, HPAF (Lan, et al. (1990) J.Biol. Chem. 265:15294-15299) (See Table 3).

                  TABLE 2                                                         ______________________________________                                        Monosaccharide composition of affinity purified                               HCA from ascites fluid.                                                       Monosaccharide  Relative Amount                                               ______________________________________                                        Fucose          1                                                             GalNAc          4.2                                                           GlcNAc          3.1                                                           Galactose       7.7                                                           Mannose         7.1                                                           NAc neuraminic acid                                                                           4.6                                                           ______________________________________                                    

FIG. 17 shows a comparison of the amino acid composition of HCA withMUC1 demonstrating clear differences. Similarly, FIG. 18 shows acomparison of the amino acid composition of HCA with TF antigen (Samuel,et al. U.S. Pat. No. 5,110,911) demonstrating that these antigens varygreatly in their constituent amino acids.

                                      TABLE 3                                     __________________________________________________________________________    Comparison of amino acid compositions of tumor                                cell sialomucins                                                              Amino acid                                                                          EPGN MUC1 ASGP                                                                              TAG72                                                                              Epit                                                                              Hupan                                                                              HC-Ag                                       __________________________________________________________________________    Serine                                                                              230  230  161 177  160 122  102                                         Threonine                                                                           153  126  175 117  69  135  36                                          Gln + Glu                                                                           101  52   134 99   141 10   146                                         Asn + Asp                                                                           49   74   58  45   91  45   82                                          Leucine                                                                             32   52   60  36   43  27   145                                         Alanine                                                                             101  77   57  54   102 161  66                                          Glycine                                                                             119  54   89  154  175 89   234                                         Valine                                                                              39   77   62  38   45  60   37                                          Proline                                                                             52   92   72  99   0   190  0                                           Lysine                                                                              27   18   22  37   51  8    48                                          Isoleucine                                                                          16   29   23  46   18  9    37                                          Arginine                                                                            29   18   35  78   36  42   43                                          Phenylala.                                                                          13   30   16  13   18  10   0                                           Tyrosine                                                                            8    29   8   13   18  10   0                                           Methionine                                                                          6    7    7   6    4   3    0                                           Histidine                                                                           26   27   22  32   27  45   24                                          __________________________________________________________________________

EXAMPLE 23 Specificity study of anti-epiglycanin AE-3

To determine the binding specificity of anti-epiglycanin AE-3,microtiterplate wells coated with epiglycanin were incubated with amixture of AE-3 antibody and native HCA or HCA that had been incubatedwith either: 10 mM NaIO₄ for 30 min. at 20° C., neuraminidase (Vibriocholerae), endo-α-N-acetylgalactosaminidase (F), Trypsin, or Pronase.The results of these experiments indicated that the antibody AE-3 bindsto a carbohydrate-containing epitope or to an epitope that requires acertain carbohydrate structure in order to maintain antibody bindingconformation.

FIG. 19 shows the competition of binding between epiglycanin andmonoclonal anti-epiglycanin antibody AE-3 by HCA or modified HCAisolated from human ascites fluid. Wells coated with epiglycanin wereincubated with a mixture of AE-3 antibody and native HCA (×) or HCA thathad been incubated with either: 10 mM NaIO₄ for 30 min. at 20° (),neuraminidase (▴), endo-α-N-acetylgalactosaminidase (♦), Trypsin (⋄), orPronase (∘).

Treatment of isolated glycoproteins or epiglycanin with TPCK-trypsin,pronase, neuraminidase, O-glycanase or periodate followed by testing ofantigenic activity indicated that the anti-epiglycanin antibodies boundto Galβ(1→3)GalNAc-containing epitopes on the glycoproteins.

EXAMPLE 24 Comparison of the specificity of anti-epiglycanin antibodiesand other tumor specific antibodies.

In the experiment shown in FIG. 20, the co-expression andcross-reactivity between the anti-epiglycanin antibodies, G-1 and AE-3,is compared to antibodies against TF-specific antibody, HH8 (Clausen, etal. (1988) Mol. Immunol. 25:199-204), sialosyl-Tn-specific antibody,TKH2 (Kjeldsen, et al. (1988) Cancer Res. 48:2214-2220), Tn-specificantibody, TKH6 (Clausen and Hakomori (1989) Vox. Sang. 56:1-20) and anantibody specific for blood group A antigen, AH16 (Clausen and Hakomori(1989, supra).

A microtiterplate coated with epiglycanin (4-61) was incubated withdilutions of mouse monoclonal antibodies followed by incubation withgoat-anti-mouse Ig conjugated with alkaline phosphatase.

FIG. 20 shows that there is negligible cross-reactivity between theanti-epiglycanin antibodies G-1, AE-3 and the antibodies HH8, TKH2, TKH6or AH16, indicating that the anti-epiglycanin antibodies bind to analtogether different epitope than HH8, TKH2, TKH6, and AH16.

In the experiment shown in FIG. 21, the inhibitory activities ofepiglycanin and the disaccharide Galβ(1→3) GalNAc (TF-disaccharide) werecompared with respect to ability to inhibit the binding between AE-3 andepiglycanin. A microtiterplate was coated with epiglycanin (4-61),blocked with BSA and incubated with a mixture of anti-epiglycanin AE-3and inhibitor. The plate was then incubated with goat anti-mouseIgM-alkaline phosphatase. The TF-disaccharide was observed to inhibitthe binding between epiglycanin and AE-3 at very high concentrations.However, the inhibitory activity of epiglycanin was about 100 milliontimes higher than that of the TF-disaccharide indicating thatepiglycanin and TF-disaccharide are not cross-inhibitory.

EXAMPLE 25 Diagnosis of human carcinoma

Using the immunoassays of the invention, it is possible to detectcarcinomas and determine the stage of disease. Using an anti-epiglycaninmonoclonal antibody in the competitive binding assay described above,HCA can be identified in the sera of advanced cancer patients.

The competitive binding immunoassay of the invention was carried outusing the HCA-specific monoclonal antibody, AE3. In a study performedwith sera from 23 Stage four carcinoma patients and 70 normal patients,the levels of HCA in the sera from the Stage four patients were elevatedrelative to the levels in normal sera. The sensitivity proportion ofStage four patients with an abnormal (elevated) test values! was foundto be 88% when the specificity (proportion of normal patients withnormal test values) was held at 95%, demonstrating that the AE3-basedimmunoassay of the invention differentiates between patients withmetastatic cancer and those without disease.

The inter-assay coefficient of variation (standard deviation divided bythe mean) was computed using the 70 normals. The coefficient ofvariation was computed across days within subjects to estimate assayreproducibility. Including all normals, the co-efficient of patientvariability was 4.0%. The coefficient of variation was then calculatedacross subjects to estimate subject variations; this co-efficient ofvariability was 0.6%. These values are indicative of an assay with ahigh level of reproducibility.

Other embodiments are within the following claims.

What is claimed is:
 1. Antibody specific for human carcinoma antigen(HCA), said antibody being less reactive with mouse epiglycanin thanwith HCA.
 2. Antibody that binds HCA, wherein said antibody is producedby administering to a mammal an immunogen selected from the groupconsisting of: HCA, immunodeterminant-containing fragments of HCA, andanti-HCA-binding idiotypic antibodies; and wherein said antibody is lessreactive with mouse epiglycanin than with HCA.
 3. The antibody of claim1 wherein said antibody is a monoclonal antibody.
 4. The antibody ofclaim 1 wherein said antibody binds to Galβ(1→3)GalNAc.
 5. Idiotypicantibody that binds HCA-binding antibody.
 6. The antibody of claim 2wherein said antibody is a monoclonal antibody.
 7. The antibody of claim2 wherein said antibody binds to Galβ(1→3)GalNAc.